Abstract This application is responsive to the PAR-15-284 (Extracellular Vesicles and Substance Abuse). Cocaine self-administration significantly reduces glutamate transporter GLT1 protein expression and impairs uptake of extracellular glutamate. Glutamate transporter GLT1 is the physiologically dominant glutamate transporter in the mammalian central nervous system (CNS). GLT1 is selectively and abundantly expressed in astrocytes after postnatal development. They are typically concentrated on the plasma membranes of peri-synaptic astroglial processes where they tightly control extracellular glutamate levels to limit the ?spill-out?/?spill-over? of glutamate from excitatory synapses. The mechanisms for GLT1 dysregulation in cocaine (and other addictive substance) self-administration are currently unknown. Exosomes are a class of newly identified membrane vesicles (40-100nm) of endosomal origin that are secreted from cells; they contain various biomolecules, including proteins, lipids, mRNAs and microRNAs (miRNAs). Exosome-mediated intercellular signaling from neuron to glia and its physiological significance in the CNS are essentially unknown. Based on our previously published and additional preliminary results, we hypothesize that exosome- mediated transfer of mir-124 from neurons to astrocytes is altered, which underlie GLT1 dysregulation in the cocaine addiction model. In this application, we will focus on the following aims: 1) Investigate exosome and mir-124 transfer from neurons to astrocytes in the cocaine addiction model we will first examine exosome secretion dynamics from striatum neuronal cultures. we will also breed CD63-eGFPf/f with dopamine receptor D1 or D2 (Drd1 or Drd2) Cre mice that allow selective labeling of exosomes in D1+ or D2+ medium spiny neurons (MSNs). We will then examine the transfer of labeled exosomes from D1+ or D2+ MSNs to neighboring astrocytes in nucleus accumbens (NAc) during different stages of cocaine addiction in situ. We will also examine changes of mir-124 levels in astrocytes by mir-124 in situ hybridization in NAc in the cocaine model. 2) Determine whether mir-124-mediated up-regulation of GLT1 attenuates cocaine relapse- associated synaptic activation we will perform stereotaxic injection to deliver mir-124 into NAc core during cocaine self-administration and test whether exogenously delivered mir-124 is able to prevent GLT1 loss and attenuate enhanced synaptic activation on MSNs in NAc during cocaine relapse. In summary, this study will investigate alterations of exosome and mi-124 transfer from neurons to astrocytes in the cocaine addiction model. This study will provide novel insights about the patho(physiological) significance of exosome-mediated miRNA transfer in mammalian CNS, especially in understanding how dysregulation of neuron to glial signaling contributes to drug addiction and relapse. Lastly, the test of mir-124's effects on cocaine relapse-associated synaptic activation may provide a new approach to intervene cocaine relapse.